Abstract 213: Assessing the role of X-ray radiation in combination with a DNA-PK inhibitor on cellular activity and adhesion using an <i>in vitro</i> HER2-positive breast cancer model
Authors: Mahmud Hasan, Ioanna Liatsou, George Sgouros
Journal: Cancer Research (2022)

Abstract Introduction: Breast cancer (BC) is the most common cancer for women regardless of ethnicity, and the leading cause of cancer-related deaths in women. Radiation therapy (XRT) plays a vital role in multidisciplinary treatment approaches. Although XRT has been found to reduce breast cancer recurrence and mortality rate, like all other therapeutics, XRT contains some risks. Radiation can increase the mortality rate due to the risk of ischemic heart diseases. As part of an overall effort to understand the interaction between different types of radiations (e.g., radiopharmaceutical therapy (RPT) with β or α-particle emitters) and DNA double-strand break repair (DSB) repair pathways, we observed that impairment of DNA DSB by DNA-PKcs inhibitor (NU7441) fragmented HER2-positive BC spheroids in the presence of XRT. This could be possibly due to the modulation of cellular adhesion protein mediated by DNA-PK inhibition. In this study, we aim to assess the spheroids fragmentations and also measure the cell viability and levels of a cell adhesion protein, β1-integrin in irradiated HER2-positive monolayer cells, to get a mechanistic view for XRT and NU7441 combinatorial action so as to compare with β- or α-emitter RPT. Methods: In brief, NT2.5 BC spheroids were formed in agarose-coated 96-well plates. For both spheroids and monolayer cells, half of the media was replaced with fresh media containing 1 nM-5μM of NU7441 every other day. A colorimetric cell viability kit was used to measure cell viability and western blot analysis was performed to measure the β1-integrin level, which was normalized against β-actin. Results: Microscopic analysis suggested that XRT increased spheroid fragmentations in the presence of NU7441 compared to the XRT alone. Interaction between XRT and NU7441 (6.68% of the total variation, P&amp;lt;0.0001) was found for the cell viability data, where XRT (74.07% of the total variation, P&amp;lt;0.0001) and NU7441 (5.07% of the total variation, P&amp;lt;0.0001) both regulated cell viability on their own. Western blot data suggest that β1-integrin level was reduced for 8 Gy XRT and 5 μM NU7441 combination compared to the no XRT and vehicle combination. Moreover, 2-way ANOVA analysis revealed that XRT plays a role to regulate the β1-integrin level in NT2.5 monolayer cells (57.61% of the total variation, P&amp;lt;0.0001). Conclusion: XRT increased BC spheroids fragmentations when DNA damage was inhibited by a small molecule inhibitor. XRT and NU7441 both regulated NT2.5 cell viability with an interaction between them. Furthermore, XRT reduced β1-integrin level in monolayer NT2.5 cells. The XRT and NU7441 combination may induce fragmentations by decreasing cell viability and adhesion proteins. Future studies assessing the mechanisms of XRT and DNA PK inhibitor in BC spheroids and in vivo are warranted to confirm these results. Citation Format: Mahmud Hasan, Ioanna Liatsou, George Sgouros. Assessing the role of X-ray radiation in combination with a DNA-PK inhibitor on cellular activity and adhesion using an in vitro HER2-positive breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 213.